1. Technical Field to Which the Invention Belongs
The present invention relates to an assay method of a biological substance generating a reaction product ammonia in a sample in an accurate manner, comprising preliminarily allowing ammonia present in the sample to be consumed up; and the invention also relates to an ammonia elimination liquid reagent for the assay method, wherein use is made of a thermo-resistant isocitrate dehydrogenase with excellent stability at alkaline pHs suitable for the ammonia elimination liquid reagent as a conjugative enzyme.
2. Prior Art
Generally, detection of urea, creatinine, creatine, guanine and adenosine and the like in biological samples such as urine and serum, as well as assay of the activities of various enzymes relevant to these substances, is routinely conducted. For the detection of these substances and during reactions with the enzyme, ammonia once generated is converted to glutamic acid via glutamate dehydrogenase (sometimes abbreviated as GLD hereinbelow), to assay the NADPH decrease through the conjugation reaction: reduced nicotineamide adenine dinucleotide phosphate (NADPH)xe2x86x92nicotineamide adenine dinucleotide phosphate (NADP+). The assay was conducted at 340 nm.
In the reaction system, ammonia is assayed as a reaction product, so ammonia primarily present in a sample is also assayed and included in the resulting assay value. Therefore, these biological substances have hardly been accurately assayed. Hence, ammonia present ina sample is allowed to react with 2-oxoglutaric acid via GLD at a preliminary process, so that the ammonia is converted to glutamic acid; the reaction system for the conversion of ammonia to glutamic acid involves the change NAD(P)Hxe2x86x92NAD(P)+, so the reverse reaction NAD(P)+xe2x86x92NAD(P)H is needed to resume NAD(P)H. In this case, a conjugative reaction can be induced, with a substrate isocitric acid, isocitrate dehydrogenase and a metal ion such as magnesium ion or manganese ion. The reaction scheme is shown in FIG. 1.
The present inventors have invented a liquid ammonia elimination reagent for the elimination of such ammonia and have submitted the patent application thereof (JP-A-9-295). A thermo-resistant isocitrate dehydrogenase derived from genus Sulfolobus is used in the ammonia elimination reagent designed by the inventors. With the enzyme, however, high-level endogenous ammonia cannot thoroughly be eliminated within a given period of time (a reaction time of about 5 minutes for automatic analyzers), when a biological substance generating ammonia as a product, for example urea nitrogen, is to be assayed directly in a sample of intact urine with no dilution. Additionally, the enzyme should be used for conventional enzyme systems, namely enzyme systems essentially requiring NADPH as a reduced coenzyme, so the enzyme is disadvantageous economically and in terms of solution stability. Hence, the enzyme is not yet sufficiently satisfactory.
In recent years, a great number of laboratory test reagents in freeze-dried powers are likely to be replaced with those in solutions. The reason is that the procedure for dissolving powdery reagents in solutions prior to each use can thereby be skipped, leading to great reduction of such burdensome works on sites.
From the standpoints of higher stability in solution under storage and cost, it is needed to use NADH in place of a reduced coenzyme NADPH having been used in conventional ammonia elimination liquid reagents. However, many of isocitrate dehydrogenase species have been in combination uniquely with NADPH, so NADH has never successfully been used therefor. In other words, isocitrate dehydrogenase species of NADP+ type have essentially been used for conventional NADPH- type systems, using for example GLD.
As has been described above, conventional ammonia elimination liquid reagents are mostly in combination with NADPH, because the use of NADH therefor is absolutely never possible. It has been known a unique yeast-derived isocitrate dehydrogenase of NAD+ type. Because the enzyme is extracted from yeast only through various instrumental processes, the enzyme is hardly available at low cost; additionally, the enzyme can never satisfactorily be kept under storage in a solution state, so the enzyme can never be used for NADPH-type systems.
In such technical circumstances, the inventors have attempted to develop a novel ammonia elimination liquid reagent using a thermo-resistant isocitrate dehydrogenase, with responsiveness to NAD and responsiveness to NADP whereby the reagent can be incorporated into conventional systems and with long-term storability at alkaline pHs.